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In-Depth Information
ef
ciency of kinesin? What is the structure and biochemical complement of the rate-
limiting step during processive motility? How often do backward steps occur and
what is their signi
cance and biochemical pathway? Do single-headed kinesins
move processively in vitro or in a cell? Do multiple kinesin motors cooperate during
motion of a cargo? Some of these questions may be answered by single-molecule
imaging.
3.3.6
Dyneins
Axonemal dynein is the molecular motor that bends and waves eukaryotic cilia and
flagella. Cytoplasmic dynein helps to position the Golgi complex and other
organelles in the cell, transports vesicular cargos derived from endoplasmic
reticulum, endosomes, and lysosomes, and takes part inmovement of the chromo-
somes and the mitotic spindle in mitosis [144]. Dynein powers retrograde axonal
transport and its disruption causes neurological disease [145]. Biophysical and
molecular biological studies of dynein lag behind those of myosin and kinesin
because the large modular nature of dynein complicates its function. Purifying
dynein from cells while retaining full activity and manufacturing it within heterol-
ogous expression systems are dif
cult. But progress is being made on these
fronts [146
-
148].
Questions arise directly from the structure of cytoplasmic dynein shown in
Figure 3.8: how is the nucleotide state in the active site (AAA domain 1) commu-
nicated to the microtubule-binding domain on the stalk, 15 nm away? What
structural change accomplishes the working stroke? Cryo-electron micro-
graphs [149] and ensemble FRET studies [150] have shown that the stalk and stem
adopt different dispositions relative to the nucleotide-binding ring at various
ATPase intermediates, simulated with nucleotide analogs. These results suggest
a hypothetical rotation of the ring relative to the stalk or stem which could drive
microtubule motion.
Processivity of cytoplasmic dynein was suggested from motility experiments with
latex beads coated with dynein at very low motor densities [151]. When beads were
placed on a microtubule using an optical trap, the likelihood of binding and of
moving was linearly proportional to the probability that one active motor was located
on the bead within interaction range of the microtubule [151]. Processivity of dynein
molecules requires at least two heads [148, 152], but compared to kinesin, the
motility of the dynein dimer is more
flexible, varying in step distance, and direction,
including sideways and backward steps. Dynein also shows strong thermally-driven
diffusive motion along microtubules even when the normal ATPase activity is
blocked [153], suggesting an electrostatic interaction with the microtubule like that
of MCAK and KIF1A.
The step size for cytoplasmic dynein was reported to vary with mechanical
load from 8 nm (the spacing of the tubulin dimers) at a resisting force of
1pN
up to 24 or 32 nm at lower loads [154]. Another study, however, found mostly 8-nm
